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METU CHEMICAL ENGINEERING DEPARTMENT ChE420 CHEMICAL ENGINEERING LABORATORY III EXP.

35 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

OBJECTIVE The purpose of this experiment is to learn the basic principles of high performance liquid chromatography (HPLC), which is one of the most common analysis techniques to identify, purify and quantify the compounds of multicomponent systems. In this experiment, you will use an Ion Exclusion type of HPLC with Ultraviolet Spectrophotometry Detector to separate and analyze the constituents of organic solutions.

GENERAL CONCEPTS IN CHROMATOGRAPHY Chromatography is essentially a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction [1]. The chromatographic separations therefore have three distinct features: a) they are physical methods of separation; b) two distinct phases are involved; c) separation results from differences in the distribution constants of the individual sample components between the two phases. Chromatographic separations depend on a favorable contribution from

thermodynamic and kinetic properties of the compounds to be separated. Molecular interactions leading to differential affinities of solutes for two phases are associated with polar forces (dipole-dipole and H-bonding) and dispersion forces (induced dipole) [2]. Useful chromatographic separations require an adequate difference in the strength of physical interactions for the sample components in the two phases, combined with a favorable contribution from system transport properties that control the movement within and between phases. Individual compounds are distinguished by their abilities to participate in common intermolecular interactions in the two phases, which can generally be characterized by equilibrium constant, and is thus a property that can be predicted from chemical thermodynamics.

A convenient classification of chromatographic techniques can be made in terms of the physical state of the phases employed for the separation. When the mobile phase is a gas and the stationary phase is a solid or liquid, the separation technique is known as gas chromatography (GC). In GC, the analyte (the chemical constituent that is of interest in the analytical procedure) must be vaporized in order for migration through the capillary to occur. When the mobile phase is a supercritical fluid, and the stationary phase being either a solid or an immobilized liquid, the separation technique is called supercritical fluid chromatography (SFC). For GC and SFC, the dominant separation mechanisms are partitioning between bulk phases and interfacial adsorption. Transport of solute zones in column chromatography occurs entirely in the mobile phase. Transport is an essential mechanism of the chromatographic system since the common arrangement for the experiment employs a sample inlet and detector at opposite ends of the column with sample introduction and detection occurring in the mobile phase. In a typical elution chromatography, which is the most convenient method for analysis (and also used in this analysis), the mobile and stationary phases are normally at equilibrium. The sample is applied to the column as a discrete band by injection, and sample components are successively eluted from the column diluted by the mobile phase. The mobile phase must compete with the stationary phase for the sample components, and for a separation to occur, the distribution constants for the sample components resulting from the competition must be different.

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY Modern liquid chromatography is dominated by the use of inorganic oxides as stationary phases with organic functional groups chemically bonded to their surface, known as bonded phases, and to a lesser extent, porous polymers. High-performance liquid chromatography (HPLC) is unquestionably the most widely used of all of the analytical separation techniques due to its sensitivity, its ready adaptability to accurate quantitative determinations and its suitability for separating nonvolatile species or thermally fragile ones [1-3]. About an order of magnitude smaller particle sizes with respect to open column LC, allow HPLC to attain higher column efficiencies, while higher pumping pressures are required to overcome the large back-pressures due to the smaller packing particles. HPLC has a widespread applicability to substances that are of prime interest to industry, to many fields of

science, and to the public. Examples of such materials include amino acids, proteins, nucleic acids (DNA and RNA), hydrocarbons, carbohydrates, drugs, terpenoids, pesticides, antibiotics, steroids, metal-organic species, and a variety of inorganic substances. Types of Liquid Chromatography Liquid Chromatographic modes can be categorized in five different groups according to their retention mechanism as can be seen in Figure 1:

Liquid Chromatography

Adsorption Competition between liquid mobile phase and solid adsorbent

Partition Competition between liquid mobile phase and liquid stationary phase Size Exclusion Molecular sieving Affinity Lock and Key mechanism

Ion Exchange Competition between liquid mobile phase and ionic stationary phase

Figure 1: Liquid Chromatography Modes The mechanism of interaction between the analyte molecules and the stationary phase particles can be categorized as (a) adsorption interactions, (b) partitioning, and (c) ionexchange. When the stationary phase is a solid, and interfacial adsorption is the dominant separation mechanism, the technique is referred to as liquid-solid chromatography (LSC). Polar silica, alumina or metal oxide particles are used as the stationary phase, while the solvent systems used are typically mixtures of relatively nonpolar liquids. The combination of a polar stationary phase and a non-polar mobile phase is called normal-phase HPLC, where the analyte is retained due to the interactions between its polar functional groups and the polar groups on the packing surface. Analytes elute in the order of increasing polarity, i.e. the least polar compound will elute from the column first, followed by each compound in the order of increasing polarity. This technique is preferred to separate analytes with low to intermediate polarity, and sufficient solubility in nonpolar solvents. Partition chromatography is based on solubility interactions between the analyte and the stationary phase. In most cases, the stationary phase is a porous silica particle to which an organic moiety (a functional chemical group) has been bonded. The organic substituent serves effectively as a bound liquid that can interact with analyte molecules. This type of stationary phase is called a bonded phase packing. The most popular bonded phase packings are based

on C8 or C18-alkyl chains. This creates a non-polar stationary phase. Solvent systems used with these stationary phases are typically mixtures of polar liquids such as water, methanol, and acetonitrile. The use of a non-polar stationary phase and a polar mobile phase is called reversed-phase (RP) HPLC, which has become the most popular separation mode in modern liquid chromatography. Since the analytes are attracted to the surface by their nonpolar functional groups, they elute (to be washed out of the column, within the mobile phase) in the order of increasing polarity, i.e. the most polar compound will elute from the column first, followed by each compound in the order of decreasing polarity. Reverse phase chromatography is preferred for separating compounds with moderate to high polarity, and sufficient solubility in polar solvents. Ion Exchange Chromatography (IEC) employs either a porous polymeric stationary phase or silica substrate with immobilized acidic or basic functional groups attached as ionic sites. The dominant separation mechanism is based on electrostatic interactions between ions in the mobile phase and those on the stationary phase, which can allow an ion exchange reaction to take place. The magnitude of the reaction equilibrium constant represents the affinity of an analyte ion for the stationary phase. The mobile phase is also an aqueous solution of ionic species, which compete with the analyte for exchange sites on the stationary phase. Affinity chromatography (AC) is a type of liquid chromatography that makes use of biological-like interactions for the separation and specific analyses of sample components [4]. The stationary phase is a solid with immobilized molecular recognition sites which can establish a specific interaction with a specific solute molecule; for example, the immobilized molecule may be an antibody to some specific protein. In this specific example, when a solute containing a mixture of proteins is passed by this molecule, only this specific protein can react with this antibody, and so binds to the stationary phase. This protein can later be extracted by changing the ionic strength or pH of the mobile phase. If the stationary phase is a solid with a controlled pore size distribution, and solutes are separated by size differences, i.e. molecular sieving, then the technique is called sizeexclusion chromatography (SEC). On the contrary to the previous chromatography modes explained above, this type of chromatography which is also known as gel permeation or gel filtration, lacks an attractive interaction between the stationary phase and the solute. Since the pores are small and exclude the larger solute molecules, only the smaller molecules can enter

the gel and thus flow through a larger volume. Therefore, larger molecules have less retention times, passing through the column at a faster rate than the smaller ones. Instrumentation for HPLC The basic components of a high-performance liquid chromatographic system is shown in Figure 2. The instrument consists of (a) eluent containers for the mobile phase, (b) a pump to move the eluent and sample through the system, (c) an injection device to allow sample introduction, (d) a column(s) to provide solute separation, (e) a detector to visualize the separated components, (f) a waste container for the used solvent, and finally (g) a data collection device to assist in interpretation and storage of results [3]. The pump, injector, column, and detector are connected with tubing of narrow inner diameter. The choice of detector is based on intrinsic properties of the solute. Often more than one detector can be used to maximize sample information and conform peak identities.

Figure 2. Schematic diagram of the basic modules of an HPLC system.

The pumping system with reciprocating pistons can supply either an isocratic or a gradient elution depending on the number of pumps used in the system. In isocratic elution, the mobile phase composition is kept constant throughout the analysis, while in gradient pumping systems, more than one solvent can be used, and so the composition of the mobile phase can be altered, which requires multiple pumps. The sample injection of the system can be either a manual injection using 6-port valve, or an automated sample injection device. Two

types of detectors are widely used for monitoring separations. An absorbance detector measures the absorbance of a particular wavelength of ultraviolet light, usually tuned for aromatic compounds. The alternative is a detector that monitors changes in the refractive index of the solvents being eluted. This allows for detection of compounds that may be transparent to UV radiation. One of the most critical factors in HPLC analysis is the quality of the solvents used [3]. In addition to being particulate and contaminant free, solvents must contain minimal levels of dissolved gases. Dissolved gases come out of solution when the eluents are pumped through the system, and the probability of gases bubbling out of solution increases when two or more liquids are mixed. The microbubbles frequently become attached to check valves, and interfere with accurate flow. In addition, they can become lodged in detector cells, creating baseline instability. Therefore, the mobile phase and all the solvents must be degassed prior to their use in the instrument. The Output of Chromatography-Chromatograms The information obtained from a chromatographic experiment is contained in the chromatogram [1]. When the elution mode is used, this consists of a plot of a detector response (y-axis) as a continuous function of time or volume of mobile phase passed through the column (x-axis). The chromatogram contains a number of peaks of various sizes rising from a baseline. Information readily extracted from the chromatogram includes an identification of a samples complexity from the number of observed peaks; qualitative identification of sample components from the accurate determination of the peak positions; quantitative assessment of the relative concentration or amount of each component from their peak areas; and the characteristic physical properties of either the solute or the chromatographic system from the peak positions and profiles. The position of a peak in a chromatogram is characterized by its retention time (t R ), which is the time taken for a specific solute to reach the detector, or retention volume (V R ), which is the volume of solution that is pumped through the detector before a specific peak is eluted (Figure 3). There are four major concepts, in terms of which chromatography is described: capacity, efficiency, selectivity and resolution [3]. The capacity and selectivity of the column are variables that are controlled largely by the column manufacturer, whereas efficiency and resolution can be controlled to some extent by the operator. To obtain the best possible separation, the efficiency of the chromatographic system must be optimized in order to

minimize band broadening. The column should have the capacity to retain the solutes, and the ability to separate sample components efficiently. The capacity factor k R of a column is a direct measure of the strength of the interaction of the sample with the packing material:

where t R is the retention time and t 0 is the time taken for non-retained species to reach the detector (hold-up time).

0 0

Eqn 1

Figure 3: Chromatographic terms used to calculate column capacity The capacity factor of a column is mostly a function of the packing material, but can be manipulated to a degree by varying the solvent strength. The higher the k R , the greater is the ability of the column to retain solutes. However, this also means longer analysis times; therefore, a compromise between resolution and analysis time must be reached. A k R value between 2 and 5 represents a good balance between the analysis time and resolution. The selectivity () of chromatographic separation is a measure of the difference in retention times between two given peaks and describes how effectively the system can separate two compounds.

The selectivity of a column is primarily a function of the packing material; although the chromatographer has some control using the mobile phase or temperature. An effective way to increase is to change the composition of the mobile phase. The resolution represents the degree of separation between neighboring solute peaks. It depends on the selectivity (), efficiency (N), and capacity (k R ) of the column.

2 0 1 0

2 1

Eqn 2

Peak broadening occurs generally due to sample components interactions with the stationary phase, and the torturous path that the solute has to take through the packing material. The efficiency of a column, N, is a number that describes peak broadening as a function of retention, and can be described simply in terms of the number of theoretical plates. In plate theory, the column is considered to consist of a number of thin plates, each of which allows a solute to equilibriate between the stationary and mobile phases. The greater the number of theoretical plates, i.e., the efficiency, N, the more efficient the column is considered to be. The movement of a solute along the column is viewed as a stepwise transfer from one theoretical plate to the next. N can be defined using the half-height method as: = 5.545(
1/2

1 1 4

( 12 )

1+

Eqn 3

where W 1/2 is the peak width at the half height of the peak (in units of time) (Figure 4).

)2

Figure 4: Baseline width (W b ) and width-at-half-height (W 1/2 ) of a chromatographic peak. The efficiency can be varied by changing the physical parameters such as length, diameter, and construction material of the container of the column. It can also be varied by

changing chemical parameters such as the size of the particles constituting the packing material or the mobile phase velocity. Note: The efficiency of a column is described more realistically with the rate theory which avoids the assumption of an instantaneous equilibrium, and addresses the diffusional factors contributing to band broadening in the column, such as eddy diffusion, longitudinal diffusion, and resistance to mass transfer in the stationary phase and the mobile phase. Contribution of these factors to efficiency is represented in a Van Deemter equation, and a plot is used to determine the experimental conditions required to obtain the most efficient system [3]. References: 1- C. F. Poole, The Essence of Chromatography, 1st ed., Elsevier, Amsterdam, 2003. 2- D. A. Skoog, J. J. Leary, Principles of Instrumental Analysis, 4th ed., Saunders College Publishing, Fort Worth, 1992. 3- A. Weston, P. R. Brown, HPLC and CE: Principles and Practice, 1st ed., Academic Press, San Diego,1997. 4- D. S. Hage, Affinity Chromatography: A Review of Clinical Applications, Clinical Chemistry, 45, 5, 593-615, 1999. PRELIMINARY WORK Be prepared to answer the following questions prior to the laboratory session. What is HPLC used for? What is the main difference between an HPLC and GC? What is the basic principle of HPLC? What are the types of liquid chromatography? What are the separation mechanisms in each type? What are the most commonly used detectors in HPLC? Get familiarize with their working principles. What is the principle of ion exclusion chromatography? (Please use instrumental analysis textbooks or other sources to learn the answer). What are the basic working priciples of reciprocating piston pumps and six-port valves? Study the acid dissociation phenomena, look into the pK a values of the common strong and weak acids.

Describe the following chromatographic parameters: column capacity factor, selectivity, resolution and number of theoretical plates. How would you manipulate these parameters in a chromatographic system?

EXPERIMENTAL PROCEDURE

1. Make sure that a baseline has been attained before starting to analysis. injected, composition of the mobile phase.

2. Take readings of the mobile phase flow rate, column temperature, minimum and 3. Preparation of the mobile phase; (this will have been already prepared for the run, so you can skip this step; but you must know how it is prepared.) L of MilliQ. Filter it through 0.45 m cellulose acetate membrane filter. Degass the for the next week, the fresh solution should be prepared since microbial growth will occur in the solution and it will obviously absorb air with time. maximum pressure limits of the pumps, elution profile, sample volume that is

Prepare 8 mM of sulfuric acid solution with Millipore Q (high purity water). Put 435 l of 96-98% (w/w) sulfuric acid (as received from Merck, 5 L bottles) into 1 solution for 10-15 minutes at room temperature. Filtered and degassed mobile phase is now ready to use in HPLC. The mobile phase can be used at most 1 week;

4. To obtain the calibration curves for the standards;

The standards, oxalic acid, citric acid and malic acid will be prepared at a acid solution at a certain concentration less than 300 ppm. These standards will

pipette (1ml) and sample vials for the preparation of these standards.

ppm, and different than the concentration of the first set of standards. Use a glass

allow you to detect the retention times of each acid. Next, prepare three different standards, each with all three acids at a certain concentration again less than 300

concentration of 500 ppm (stock solutions). For standard preparation, mobile

phase solution should be used. Prepare different standards, each with one type of

5. Press single injection and upload the method file (recently uploaded in the manual injection.

software). Also upload a data file which represents the name of the single sample that will be analyzed in the device. Fill the sample volume as 20 L and press OK. A small box is going to appear which specifies that the device waits for the You will not be using an auto-sampler so you have to perform injections manually

through the injection port and wait until each analysis will be completed. Before each injection make sure that you rinse your syringe (stainless steel syringe) with mobile phase to remove any contaminants. Slowly and completely fill the syringe not contain any bubbles. Any air bubble can cause damage for the device. These with your sample. Before injection, make sure that the sample in the syringe does

syringes are non-dispensable and expensive. So please pay attention while using it. Before injection, the injection port should be in load mode (left). Put the syringe (right), at the same time you will see the running alert on the monitor. procedure for the samples with unknown compositions. complete the post-run calculations for your report. to the injection port gently. Make the injection. After you make the injection of 50 l and remove the injector from the port, you will turn the port to the inject mode

6. After you complete this procedure for all of your standards, repeat the same 7. Obtain your runs data report. Make sure you have all the data you need to
DATA SHEET

Initial readings of the main menu Analysis Time: Flow rate of mobile phase: Column temperature: Injection volume: Pressure limits of the pump: UV absorbance wavelength: Composition of the mobile phase:

STANDARDS 1 2 3 4 5 6

Compound

Concentration Retention (ppm) time

Peak Area

All three acids All three acids All three acids

UNKNOWN

NonNonretained retained species, species, Rt Area

R t, 1

Area1

R t, 2

Area2

R t, 3

Area3

w h1

w h2

w h3

CALCULATIONS 1. Construct the calibration curve for each species. Use least squares procedures to 2. Determine the concentration of the components in the unknown solution. compute the slope, y-intercept, r2, and standard error of estimate. a. Capacity factor of the column for each species 3. Choose one of your unknown samples and calculate:

b. Selectivity of the column; i.e., selectivity for citric acid over oxalic acid, and c. resolution of the two sets of neighboring peaks plates for each species d. efficiency of the column for each species: i.e., the number of theoretical for malic acid over citric acid

Please discuss the results of your calculations. Your discussions should include your comments about the efficiency and reliability of this analysis, and if you have any suggestions to improve it.

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